WO2017055844A1 - Porc génétiquement modifié - Google Patents

Porc génétiquement modifié Download PDF

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Publication number
WO2017055844A1
WO2017055844A1 PCT/GB2016/053025 GB2016053025W WO2017055844A1 WO 2017055844 A1 WO2017055844 A1 WO 2017055844A1 GB 2016053025 W GB2016053025 W GB 2016053025W WO 2017055844 A1 WO2017055844 A1 WO 2017055844A1
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Prior art keywords
rela
nucleic acid
genetically
swine
introgressed
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PCT/GB2016/053025
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English (en)
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Christopher Bruce Alexander Whitelaw
Simon Geoffrey Lillico
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The University Court Of The University Of Edinburgh
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Priority to JP2018535263A priority Critical patent/JP2018529384A/ja
Priority to CA3000304A priority patent/CA3000304A1/fr
Priority to US15/762,363 priority patent/US20180271068A1/en
Priority to EP16778437.0A priority patent/EP3355690A1/fr
Priority to CN201680057129.3A priority patent/CN109068620A/zh
Priority to KR1020187011667A priority patent/KR20180054838A/ko
Priority to AU2016332623A priority patent/AU2016332623A1/en
Publication of WO2017055844A1 publication Critical patent/WO2017055844A1/fr
Priority to HK19101782.9A priority patent/HK1259412A1/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/108—Swine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/02—Animal zootechnically ameliorated
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses

Definitions

  • the present invention relates to genetically-edited swine comprising an introgressed heterologous nucleic acid sequence in the RELA gene.
  • Fever Virus in contrast to present-day pig species found in Africa.
  • warthog Phacochoerus africanus
  • domestic pig RELA 6 we have earlier identified three amino acid differences between warthog (Phacochoerus africanus) and domestic pig RELA 6 .
  • WO2014/041327 describes genome editing of pigs by creating indels via NHEJ after DNA cleavage by ZFNs and TALENs, but does not describe allele introgression.
  • the present inventors have achieved success in carrying out allele introgression in the RELA gene of pigs. This invention thus overcomes considerable uncertainty to complete this landmark achievement.
  • the inventors have described for the first time the introgression of a heterologous nucleic acid into the swine genome; in particular, they have introgressed a complete haplotype of the warthog RELA allele into the domestic pig RELA gene.
  • the introgression of such a haplotype into the pig genome is a remarkable achievement, and the resultant pigs are of considerable commercial interest.
  • a genetically-edited swine comprising an introgressed heterologous nucleic acid sequence in the RELA gene.
  • the swine is a pig, and more preferably a domestic pig.
  • the RELA protein is a predominant component of the NFkappaB heterodimeric transcription factor.
  • genetic editing which alters the levels or activity of RELA will directly affect NFkappaB dependent cell activities, in particular transcription from NFkappaB induced genes.
  • NFkappaB is a key effector of animal responses to various stresses, including infection. Genetically-edited animals with altered RELA expression or activity will therefore react differently to their non-edited counterparts in response to biological stresses or insults, such as infection, chronic and/or autoimmune diseases.
  • the introgressed heterologous nucleic acid sequence comprises a heterologous RELA allele.
  • the introgressed heterologous allele comprises a trans- species heterologous RELA allele.
  • the introgressed heterologous allele comprises a trans-genus heterologous RELA allele.
  • the introgressed heterologous allele converts a wild-type RELA allele to a heterologous RELA allele, more preferably a trans-species heterologous RELA allele or a trans-genus heterologous RELA allele, and suitably the warthog RELA allele.
  • an allele of the RELA gene (which can include regulatory and non-coding sequences) present in an animal (e.g. a domestic pig) can be 're-written' via introgression such that a different allele is present - in many cases this may involve changes to only small number of bases. This can be done in a completely 'clean' manner, i.e. no footprint or other trace of the editing event is left behind, and the only changes made to the genome are those required for the desired allele conversion. In this way, for example, a RELA allele that is naturally found in one species, can be introduced into a population (e.g. species) in which the allele is not present. This approach is very different from conventional transgenesis in which genes (and often selectable markers) are inserted, moved or disrupted, but wherein some form of footprint, in the sense of genomic disruption, results.
  • the heterologous allele of the present invention is not formed by a deletion, inversion, or other such random or poorly-controlled edits which are the typical result of non-homologous end joining (NHEJ).
  • the introgressed heterologous allele is typically introgressed by allele conversion via homology directed repair (HDR) of a site-specific nuclease (SSN) induced double stranded break (DSB) in the genomic DNA at or near the locus of said allele based upon a template nucleic acid (typically DNA) sequence comprising the sequence of the heterologous allele.
  • HDR homology directed repair
  • SSN site-specific nuclease
  • DSB induced double stranded break
  • the genetically-edited swine comprises an introgressed RELA allele that differs from the wild-type RELA allele sequence by changes in two or more bases.
  • the swine thus preferably comprises a introgressed RELA haplotype.
  • the haplotype is preferably introgressed via a single editing event.
  • the haplotype can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more base changes compared with the wild-type RELA sequence.
  • the genetically-edited swine comprises an introgressed nucleic acid in the RELA gene which is 50 or more bases in length, suitably 100 or more, 150 or more, 200 or more, 250 or more, 500 or more, or 1000 or more, or 1500 or more bases in length.
  • the distance spanning the 2 or more bases which are altered can be 50 or more bases, optionally 100 or more, 150 or more, 200 or more, 250 or more, 500 or more, or 1000 or more bases.
  • all cells of the genetically-edited swine contain the introgressed heterologous nucleic acid sequence. This can be achieved, for example, by modifying the single-cell zygote and raising a swine from the zygote.
  • the introgressed RELA allele preferably changes the sequence of the RELA protein encoded by the RELA gene. It is generally preferred that the introgressed RELA allele results in an alteration to the coding region (exons) of the RELA gene, i.e. corresponding to the cDNA sequence set out in SEQ ID NO 15.
  • the introgressed RELA allele thus preferably results in a change of one or more amino acids relative to the wild-type domestic pig RELA amino acid sequence shown in SEQ ID NO 16.
  • the introgressed RELA allele changes sequences in the region of the RELA gene which encodes the transactivation domain of RELA.
  • such domains extend from amino acid 431 to 553 of the wild-type RELA protein sequence (unless otherwise stated, nucleic acid and amino acid numbering is with reference to the wild-type Sus scrofa RELA cDNA or protein sequences).
  • Transactivation domain 2 extends from amino acid 431 to 521 and transactivation domain 1 extends from amino acid no 522 to the C-terminus of the protein at amino acid 553. More preferably, the introgressed RELA allele changes the region of the RELA gene which encodes amino acids 448 to 531 of RELA.
  • the genetically-edited swine is a pig that comprises an introgressed trans-species allele of the RELA gene.
  • the pig comprises an introgressed heterologous nucleic acid sequence which converts the domestic pig RELA sequence to the sequence of a trans-species allele.
  • the pig comprises an introgressed trans-species allele of the RELA gene from a swine species outside of the genus Sus.
  • the pig comprises an introgressed trans-species allele of the warthog RELA gene, and in particular an allele of the sequence encoding the transactivation domain of RELA.
  • the introgressed heterologous nucleic acid sequence is a sequence which is not present within the breed, sub-species, and preferably the species, of the genetically-edited swine (e.g. it is non-native to the relevant species).
  • the introgressed heterologous nucleic acid sequence suitably comprises a heterologous RELA allele that is not present in domestic pigs.
  • the swine can be heterozygous (mono-allelic) or homozygous (bi-allelic) for the introgressed RELA allele.
  • the swine is homozygous (bi-allelic) for the introgressed RELA allele.
  • the modification causes a change in the amino acid located at one or more of the following amino acids of RELA:
  • amino acids at two or more of these sites are altered.
  • amino acids at all three of the sites are altered.
  • the modifications may suitably result in the following changes in the amino acids of RELA: T448A, S485P, and/or S531 P. These alterations correspond to polymorphisms that have been observed between domestic pigs and warthogs. These polymorphisms correlate with tolerance to ASFV infection in warthogs.
  • the modifications may suitably result in one of the following changes in the amino acids of RELA: T448A; S485P; S531 P; T448A and S485P; S485P and S531 P; T448A and S531 P; T448A, S485P and S531 P.
  • the swine is a pig that comprises an introgressed heterologous nucleic acid sequence which results in the following amino acid changes to the domestic pig RELA protein: T448A, S485P and S531 P.
  • T448A, S485P and S531 P are the amino acid differences between the wild-type RELA protein sequence in domestic pigs and the wild-type RELA protein sequence in warthogs. It is preferred that no amino acid changes other than T448A, S485P and S531 P are caused by the introgressed heterologous nucleic acid.
  • a genetic editing event means that these three amino acid changes are made (and no others) to a domestic pig
  • a perfect trans-genus allele conversion has occurred, i.e. perfectly converting the domestic pig RELA allele to the corresponding warthog allele in the absence of any unintended changes.
  • Warthogs contain several other polymorphisms at the nucleic acid level but they do not affect the expressed amino acid sequence and thus in the present case can be ignored.
  • the present invention thus provides a genetically-edited pig wherein the RELA gene has been edited such that it comprises the sequence as set out below (the amino acids at sites 448, 485 and 531 are shown in bold):
  • the present invention thus provides a domestic pig which has been genetically- edited such that at least a portion of the autologous RELA sequence that includes the sequences encoding S531 , T449 and S485 has been replaced by introgression (suitably via HDR of a DSB induced by a suitably targeted SSN) of a sequence which encodes the corresponding warthog (Phacochoerus sp.) RELA protein sequence.
  • the introgressed nucleic acid sequence can be identical to the warthog sequence or can be an equivalent artificial sequence comprising one or more synonymous base changes.
  • the genetically-edited swine is a pig (preferably a domestic pig) which has improved tolerance to ASFV infection resulting from the introgressed heterologous nucleic acid.
  • An animal can be said to be more tolerant to infection when the morality rate, morbidity rate, the proportion of animals showing significant morbidity (e.g. weight loss or decreased growth rate), the level of morbidity or the duration of morbidity is reduced.
  • the morbidity rate approaches 100% in naive herds.
  • the mortality rate depends on the virulence of the isolate, and can range from 0% to 100%. Highly virulent isolates can cause almost 100% mortality in pigs of all ages. Less virulent isolates are more likely to be fatal in pigs with a concurrent disease, pregnant animals and young animals.
  • the mortality rate may be as high as 70-80% in young pigs, but less than 20% in older animals. Any statistically significant reduction (e.g. 95% confidence, or 99% confidence using an appropriate test) in the mortality or morbidity between a population of genetically-edited pigs and a population of equivalent non-edited pigs when exposed to ASFV of the same virulence level (ideally the same isolate) demonstrates improved tolerance.
  • a cell nucleus, germ cell, stem cell, gamete, blastocyst, embryo, foetus and/or donor cell of a swine comprising an introgressed heterologous nucleic acid sequence in the RELA gene.
  • the cell nucleus, germ cell, stem cell, gamete, blastocyst, embryo, foetus and/or donor cell is from a domestic pig, and comprises an introgressed trans-species RELA allele, e.g. the warthog RELA allele.
  • the cell nucleus, germ cell, stem cell, gamete, blastocyst, embryo, foetus and/or donor cell is derived from a swine as set out above. Alternatively, it can be created de novo using the methods described herein.
  • the invention provides a method of producing a genetically-edited swine having an introgressed heterologous nucleic acid sequence in the RELA gene, the method comprising the steps of:
  • nuclease being adapted to
  • introducing a template nucleic acid comprising the heterologous nucleic acid adapted to introgress the heterologous nucleic acid sequence into the RELA gene, the heterologous sequence being flanked by sequences homologous to genomic RELA sequences;
  • the swine is a pig, and more preferably a domestic pig.
  • the site-specific nuclease comprises a zinc finger nuclease (ZFN), a Transcription Activator-Like Effector Nuclease (TALEN), an RNA-guided CRISPR/Cas nuclease
  • ZFN zinc finger nuclease
  • TALEN Transcription Activator-Like Effector Nuclease
  • RNA-guided CRISPR/Cas nuclease RNA-guided CRISPR/Cas nuclease
  • the site-specific nuclease comprises a pair of cooperating ZFNs, TALENs or RNA-guided CRISPR 'nickases' (e.g. having a modified Cas9 nuclease capable of cutting only one DNA strand), adapted such that DNA cutting only occurs when both members of the pair are present and form a heterodimer, which is able to cut both strands of the DNA molecule.
  • the use of a pair of cooperating ZFNs, TALENs or RNA-guided CRISPRs results in a reduction of possible off-target cutting events.
  • the site-specific nuclease comprises a pair of ZFNs.
  • the site-specific nuclease is adapted to target and cut within the region of the RELA gene encoding the transactivation domain of RELA (i.e. amino acid 431 to 553 of the wild-type RELA protein sequence) or slightly upstream or downstream thereof, e.g. within 500, 300, 200, 100, 50 or 20 bases upstream or downstream thereof. More preferably the site-specific nuclease is adapted to target and cut within region of the RELA gene which encodes amino acids 448 to 531 of RELA, or slightly upstream thereof, e.g. within 500, 300, 200, 100, 50, 20 or 10 bases upstream thereof. Preferably the site-specific nuclease is adapted to target and cut within exon 9 of the RELA gene.
  • the site-specific nuclease is adapted to target and cut upstream of the region of the RELA encoding amino acid T448, e.g. within 500, 300, 200, 100, 50 or 20 bases upstream thereof.
  • the site-specific nuclease is adapted target a suitable sequence to cut at a site lying between bases 1200 and 1341 (with reference to SEQ ID NO 15), more preferably at a site lying between bases 1250 and 1340, yet more preferably at a site lying between bases 1300 and 1340, and yet more preferably at a site lying between bases 1320 and 1340.
  • the cut site lies between bases 1332 and 1333.
  • the target site of one of a pair of cooperating SSNs is GATACTGATGAGGAC (SEQ ID NO 18) and the target site of the other of the pair of SSNs is CTCCGGGACGACGTC (SEQ ID NO 19).
  • Other target sequences could be used, and the skilled person is readily able to determine suitable target sites optimised for different SSNs.
  • the site-specific nuclease can be introduced to a cell in any suitable form.
  • the nuclease can be provided directly into the zygote as a functional protein.
  • the nuclease can be provided into the zygote in the form of a precursor or template from which the active nuclease is produced by the zygote.
  • an mRNA encoding the nuclease is introduced into the zygote, e.g. by injection. The mRNA is then translated by the cell to form the functioning protein. Using mRNA in this way allows rapid but transient expression of the nuclease within the cell, which is ideal for the purposes of genetic editing.
  • the term 'zygote' can be used in a strict sense to refer to the single cell formed by the fusion of gametes. However, it can also be used more broadly to refer to the cell bundle resulting from the first few divisions of the true zygote (this is more properly known as the morula). It is preferred that the present method is at least initiated, and preferably completed, in the zygote at the single cell stage.
  • the genetically-edited zygote can be grown to become an embryo and eventually an adult animal. If the editing event occurs in the single-cell zygote then all cells of this animal will comprise the modified RELA gene as all cells of the animal are derived from a single genetically-edited cell. If the editing event occurs after one or more cell divisions then the resultant animal will likely be a mosaic for the editing event, in that it will have some cells derived from the edited cell and some cells derived from unedited cells.
  • the method can be performed on a plurality of zygotes and the method may involve selecting zygotes in which the desired genetic modification has been achieved.
  • the template nucleic acid comprises a region including the heterologous nucleic acid sequence flanked on each side by homologous sequences.
  • the template construct can comprise a heterologous nucleic acid sequence that is, for example, 50 or more bases in length, suitably 100 or more, 150 or more, 200 or more, 250 or more, 500 or more, or 1000 or more, or 1500 or more bases in length.
  • the flanking homologous sequences can be, for example, 50 or more bases in length, suitably 100 or more, 150 or more, 200 or more, 250 or more, 500 or more, or 1000 or more bases in length.
  • the template nucleic acid comprises a region including the warthog RELA haplotype flanked on each side by homologous regions. It is important to note that a region including the warthog RELA haplotype would typically be largely homologous to the wild-type target sequence, except for changes at the necessary bases to achieve the desired edit(s).
  • the homologous region can be, for example, from 200 to 1000 bases in length, suitably from 500 to 900 bases in length.
  • the template nucleic acid comprises a region including the warthog RELA of 251 or more nucleotides in length, which comprises a nucleic acid sequence encoding the protein sequence
  • ADEDLGALLGNNTDPTVFTDLASVDNSEFQQLLNQGVPMPPHTAEPMLMEYPEAITRLVTG SQRPPDPAPTPLGASGLTNGLLP (SEQ ID NO 23) (the amino acids changes T448A, S485P and S531 P are shown in in bold) flanked by homologous regions.
  • the homologous regions can be 200 bases or longer, suitably 400 bases or longer, more preferably 600 bases or longer.
  • the template nucleic acid is double stranded.
  • the template nucleic acid is provided in a plasmid. Provision of the template in the form of a plasmid has been found to result in improved efficacy and/or efficiency of introgression.
  • the template is plasmid comprising a 251 bp region containing the warthog RELA haplotype (i.e. encoding SEQ ID NO 23) flanked by regions (homology arms) of 626 bp and 799 bp.
  • the 251 bp region contains 5 base changes to convert the domestic pig sequence to the warthog haplotype.
  • the template nucleic acid comprises one or more (preferably two or more, yet more preferably three or more) base changes compared with the corresponding genomic nucleic acid sequence at the target site for the SSN. Provision of such changes means that following HDR the target site for the SSN will be destroyed (or at least rendered sub-optimal), thus preventing or reducing re-cutting once a successful introgression has occurred.
  • a target site for the SSN is GATACTGATGAGGAC (SEQ ID NO 18)
  • the template nucleic acid comprises the sequence GATgCaGAcGAGGAC (SEQ ID NO 20) to replace the genomic sequence and prevent re- cutting by the SSN.
  • SSN target site is underlined, the cut site is in bold, and the changes are shown in lower case:
  • a method of producing a genetically-edited domestic pig having an introgressed warthog RELA haplotype comprising the steps of:
  • a pair of cooperating site-specific nucleases (suitably ZFNs), the nucleases being adapted to target the RELA gene in the region of (preferably upstream, and suitably within 20bp) of the sequence encoding T448A and introduce a double stranded break;
  • a template nucleic acid preferably a double-stranded DNA template, e.g. a plasmid
  • a heterologous nucleic acid comprising a sequence encoding a corresponding warthog RELA haplotype flanked by sequences homologous to the genomic RELA sequence of the pig
  • Fig. 2 Sequence analysis of live born piglets. The sequence of both the domestic pig and warthog encoding the three observed amino acid differences is shown above, with sequence traces from individual animals below. Inset pictures of chromosomes indicate the allelic makeup at each position in each animal (domestic pig allele - red; warthog allele - green).
  • the term "swine”, or variants thereof, as used herein refers to any of the animals in the Suidae family of even-toed ungulates including animals in the genus Sus and other related species, including the peccary, the babirusa, and the warthog.
  • the term "pig” or variants thereof as used herein refers to any of the animals in the genus Sus. It includes the domestic pig (Sus scrofa domesticus or Sus domesticus) and its ancestor, the common Eurasian wild boar (Sus scrofa). For the present purposes the domestic pig is considered to be a sub-species of the species Sus scrofa. It does not include the peccary, the babirusa, and the warthog.
  • domestic pig or variants thereof, as used herein refers to an animal of the subspecies Sus scrofa domesticus.
  • RELA gene refers to the RELA (V-Rel Avian Reticuloendotheliosis Viral Oncogene Homolog A gene, also known as the p65 gene, NCBI Gene ID: 100135665) gene, and includes both coding and non-coding regions, and also associated regulators promoter and enhancer regions.
  • introgression modifies the sequence within the RELA gene ORF, and more preferably within at least one exon.
  • site-specific nuclease refers to engineered nucleases which can be configured to cut DNA at a desired location. Such site-specific nucleases are also known as engineered nucleases, targetable nucleases, genome editing nucleases, molecular scissors, and suchlike. Examples of site-specific nucleases include zinc finger nucleases (ZFNs), Transcription Activator- Like Effector Nucleases (TALENs), the CRISPR/Cas system (CRISPR), and meganucleases, such as hybrid meganucleases.
  • ZFNs zinc finger nucleases
  • TALENs Transcription Activator- Like Effector Nucleases
  • CRISPR CRISPR/Cas system
  • meganucleases such as hybrid meganucleases.
  • heterologous allele refers to an allele which is not present in the relevant animal.
  • the heterologous allele can be naturally occurring in another species or genus, or it can be non-natural in any species (i.e. entirely artificial). Preferably the allele is naturally occurring in another species.
  • trans-species heterologous allele refers to an allele which does not naturally occur in the species of the relevant animal, but which occurs naturally in another species.
  • the heterologous allele can be naturally occurring in another species, which species may be from the same or a different genus.
  • a trans-species allele is still a 'natural allele' in the sense that it is not artificial and is found in nature, but it is introgressed to a new species to form a new animal with desired properties.
  • trans-genus heterologous allele refers to an allele which does not naturally occur in the genus of the relevant animal, but which occurs naturally in another genus.
  • the set “trans-genus heterologous alleles” is thus a subset of "trans-species heterologous alleles", i.e. wherein a trans-genus heterologous allele comes from outside of the relevant animal's genus, and not merely from outside of the relevant animal's species.
  • the RELA allele from warthogs is a trans-genus
  • heterologous allele for animals in the genus Sus, and in particular to domestic pigs.
  • haplotype refers to a linked set of DNA sequence variations (typically single-nucleotide polymorphisms (SNPs)) at a specific locus on a single chromatid of a chromosome pair.
  • SNPs single-nucleotide polymorphisms
  • a haplotype is typically a plurality of SNPs differing between one species or genus and other, which contribute to or define a heterologous allele as between the one species or genus and the other.
  • the RELA allele in the present example there are 3 amino acid changes as between domestic pigs and warthog; these changes represent the haplotype of the heterologous RELA allele as between domestic pigs and warthogs.
  • introduction refers to the introduction of a heterologous nucleic acid sequence, especially a gene or allele, from a given source into an animal, typically by rewriting or converting an existing genomic sequence. Re-writing or converting in the present invention is achieved by HDR.
  • the source of the heterologous nucleic acid sequence can be an animal from another species or genus, or it can be an artificial sequence.
  • allele introgression refers to a genetic edit which introduces an allele to the genome of an animal.
  • the allele introgression can be an "allele conversion” or “allele replacement”, or it may, for example, introduce a new gene in its entirety.
  • the allele introgression is an allele conversion or allele replacement.
  • allele conversion or "allele replacement”, or variants thereof, as used herein refers to an introgression which replaces a normal, usually 'wild-type', allele with a heterologous allele.
  • Conversion or replacement of a wild-type allele to a heterologous allele can in some cases involve alteration of the wild-type genomic DNA sequence to exactly match the DNA sequence of the heterologous allele from another animal. However, in other cases conversion or replacement may only require modification to the wild-type genomic DNA sequence such that the encoded protein matches the protein encoded by the heterologous allele (i.e. synonymous substitutions need not be made to the wild-type genomic sequence).
  • the present inventors sought to use genome editing to introduce the entire warthog RELA haplotype (which spans 251 base pairs, bearing five SNPs resulting in 3 amino acid changes) via a single nuclease-induced DSB.
  • editing- driven haplotype introgression had not been previously reported in live born animals of any mammalian species.
  • ZFNs can be engineered to induce a DSB at any genomic position.
  • nuclease design considerations were informed by the need to transfer an entire 251 bp haplotype bearing multiple SNPs.
  • the inventors conceived a strategy (Fig 1 a) in which a ZFN is engineered for a region immediately upstream of the haplotype-marked stretch, with the intention that single-sided invasion of the repair template by the upstream chromosome arm would then lead to a synthesis-dependent strand annealing-based transfer of the entire downstream haplotype to the endogenous locus.
  • a ZFN heterodimer (see Table 1 for details) was produced that binds to the region flanking 1330 to 1338 bp relative to the translational start site in the porcine RELA cDNA sequence (NM_001 114281).
  • PK15 transformed cell line established from the domestic pig, and compared genome editing efficiencies via the Surveyor/Cel-1 endonuclease assay 10 .
  • ZFNs used to create a DSB in the genome
  • such a cut can be achieved using the various other site-specific nucleases now well-known to the skilled person.
  • a suitable TALEN pair could readily be designed to target the same locus
  • the CRISPR/Cas system could also be used by providing suitable guide RNA sequences to guide either wild-type or paired 'nickase' Cas nuclease(s).
  • ZFNs are disclosed in the present examples, and ZFNs exhibited highly desirable properties, the present invention is not be restricted to the use of ZFNs.
  • ZFN technology is described extensively in the literature and, inter alia, in the following patent documents: US 6,479,626, 6,534,261 , 6,607,882, 6,746,838, 6,794,136, 6,824,978, 6,866,997, 6,933, 113, 6,979,539, 7,013,219, 7,030,215, 7,220,719, 7,241 ,573, 7,241 ,574, 7,585,849, 7,595,376, 6,903, 185, 6,479,626, 8, 106,255, 20030232410, and 20090203140, all of which are incorporated by reference.
  • ZFNs can be obtained commercially from Sigma- Aldrich (St.
  • TALENs can be obtained commercially from Thermo Fisher
  • CRISPR/Cas technology is described extensively in the literature (e.g. Cong et al. 'Multiplex Genome Engineering Using CRISPR/Cas Systems', Science, 15 February 2013: Vol. 339 no. 6121 pp.
  • CRISPR/Cas systems can be obtained commercially from Sigma-Aldrich (St. Louis, MO, US) under the CRISPR/Cas Nuclease RNA-guided Genome Editing suite of products and services, or from Thermo Fisher Scientific, Inc. (Waltham, MA, US) under the GeneArt® CRISPR branded products and services.
  • Sigma-Aldrich Sigma-Aldrich (St. Louis, MO, US) under the CRISPR/Cas Nuclease RNA-guided Genome Editing suite of products and services, or from Thermo Fisher Scientific, Inc. (Waltham, MA, US) under the GeneArt® CRISPR branded products and services.
  • embryogenesis can be achieved by the delivery of nuclease-encoding mRNA to the embryo 12 .
  • the ORFs encoding the RELA ZFNs into two distinct vectors for in vitro mRNA production (pVAX, which requires in vitro polyadenylation, and pGEM, which contains a polyA track of defined length).
  • pVAX which requires in vitro polyadenylation
  • pGEM which contains a polyA track of defined length
  • porcine zygotes with mRNA encoding the pair of ZFN and either a single stranded oligodeoxynucleotide (ssODN 16 ) or plasmid DNA bearing the warthog SNPs. Injected zygotes were transferred to recipient gilts 14 . To determine whether the nucleases drove targeted editing of pig RELA, ear notches were taken from piglets 2 days postpartum and genomic DNA was prepared. PCR spanning the target locus and sequencing of these products was used to identify either alleles bearing small indels (a result of NHEJ) or specific point mutations (a result of HDR events).
  • Piglet 367 was homozygous for 4 base changes proximal to the ZFN target site, and heterozygous for the most distal modification. This finding demonstrated that continuity of gene conversion tracks from DSB-R in mammalian cells, and indicates that the two homologs were cleaved in the early embryo followed by distinct HDR-based resolution of the break. Remarkably, piglet 563 was homozygous for all 5 base changes. Livestock breeding has enabled a continuous increase in animal productivity since animal domestication. The challenge ahead is to accelerate this improvement process to meet the demands imposed on agriculture through climate change, resource and land availability in conjunction with the increase in human population.
  • Genome editing technology has the potential to revolutionize livestock breeding 4 , and targeted gene knockout in several livestock species has been attained using multiple distinct designed nuclease platforms, including ZFNs, TAL effector nucleases, and CRISPR/Cas9 11 ⁇ 13 ⁇ 15 ⁇ 17"19 .
  • the present inventors have significantly expanded the genome editors' toolbox to include the targeted transfer of an entire haplotype. Specifically, through homology dependent repair of a ZFN-induced break using a plasmid repair template we have introgressed an allele of the RELA gene between swine species, producing live piglets both heterozygous and homozygous for the desired haplotype.
  • ZFN Design and Validation ZFNs against the indicated position of the pig RELA gene were designed and assembled using an archive of pre-validated two-finger modules as described [2] .
  • the ORFs were cloned into expression vectors harbouring enhanced obligate heterodimer forms of Fokl [20] optimized for delivery in DNA form and for production of in vitro transcribed mRNA (Vierstra et al., in press).
  • ZFN target sequences and DNA recognition helices are described in Table 1.
  • Pig PK15 cells were electroporated using ZFN-encoding DNA or mRNA as described, genomic DNA harvested 48h following electroporation, and percentage of chromatids bearing indels was measured using Surveyor / Cell as described [ 10] or deep sequencing on the lllumina platform.
  • a 96-mer ssODN was designed spanning the target site of the ZFN and containing two base changes encoding the desired T448A conversion (Fig. 1) plus a third (silent) base change to assist in preventing ZFN re-cutting of any introgressed alleles.
  • the plasmid DNA template was designed with the same three base changes as the ssODN at the ZFN target site, with additional single base changes encoding the S485P and S531 P (Fig. 1). This 251 bp central domain containing all five base changes was flanked by homology arms of 626 bp and 799 bp, 5' to the first base change and 3' to the final base change respectively.
  • Embryos were produced from Large-White gilts that were approximately 9 months of age and weighed at least 120 kg at time of use. Super-ovulation was achieved by feeding, between day 11 and 15 following an observed oestrus, 20 mg altrenogest (Regumate, Hoechst Roussel Vet Ltd) once daily for 4 days and 20 mg altrenogest twice on the 5th day. On the 6th day, 1500 IU of eCG (PMSG, Intervet UK Ltd) was injected at 20:00hrs. Eighty-three hours later 750 IU hCG (Chorulon, Intervet UK Ltd) was injected. Donor gilts were inseminated twice 6 hours apart after exhibiting heat generated following super-ovulation.
  • Embryos were surgically recovered from mated donors by mid-line laparotomy under general anaesthesia on day 1 following oestrus into NCSU-23 HEPES base medium. Embryos were subjected to a single 2-5pl cytoplasmic injection of the pVAX single mRNAs at 2 ng/ ⁇ or 4 ng/ ⁇ with ssODN or plasmid template respectively. Recipient females were treated identically to donor gilts but remained un-mated. Following ZFN injection, fertilized embryos were transferred to recipient gilts following a mid-line laparotomy under general anaesthesia. During surgery, the reproductive tract was exposed and embryos were transferred into the oviduct of recipients using a 3.5 French gauge tomcat catheter. Litter sizes ranged from 1-13 piglets.
  • Genotyping Genomic DNA was prepared from ear biopsy taken from piglets 2 days postpartum. PCR amplification with AccuPrime HiFi was conducted with primers oSL1 (gggtacaaagaggggtgagg - SEQ ID NO 13) which binds out-with the 5' homology arm encoded by the plasmid and oSL2 (ctagctctgccctttccaga - SEQ ID NO 14) which binds within the 3' homology arm of the plasmid. Cycling was 95 °C for 120 seconds then 40 cycles of 94 °C for 30 seconds, 59 °C for 30 seconds and 68 °C for 90 seconds, followed by primer extension of 68 °C for 5 minutes. Purified PCR products were directly sequenced.

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Abstract

La présente invention concerne un porc génétiquement modifié comprenant une séquence d'acides nucléiques hétérologues introgressée dans le gène RELA. La présente invention concerne, plus précisément, un porc génétiquement modifié comprenant un allèle de phacochère introgressé dans le gène RALA des porcs domestiques. La présente invention concerne également des procédés de production d'un tel porc, et des cellules dérivées d'un porc ayant de telles séquences introgressées.
PCT/GB2016/053025 2015-09-29 2016-09-29 Porc génétiquement modifié WO2017055844A1 (fr)

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CA3000304A CA3000304A1 (fr) 2015-09-29 2016-09-29 Porc genetiquement modifie
US15/762,363 US20180271068A1 (en) 2015-09-29 2016-09-29 Genetically-Edited Swine
EP16778437.0A EP3355690A1 (fr) 2015-09-29 2016-09-29 Porc génétiquement modifié
CN201680057129.3A CN109068620A (zh) 2015-09-29 2016-09-29 基因编辑的猪
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WO2014041327A1 (fr) * 2012-09-12 2014-03-20 The University Court Of The University Of Edinburgh Animal génétiquement modifié
WO2015030881A1 (fr) * 2013-08-27 2015-03-05 Recombinetics, Inc. Introgression efficace d'allèle non-méiotique

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SIMON G. LILLICO ET AL: "Mammalian interspecies substitution of immune modulatory alleles by genome editing", SCIENTIFIC REPORTS, vol. 6, 22 February 2016 (2016-02-22), pages 21645, XP055325576, DOI: 10.1038/srep21645 *

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